Under the Microscope

Pneumocystis canis in dogs

Elizabeth RalphA, George ReppasB, Catriona HallidayC, Mark KrockenbergerD and Richard MalikE,F AAnimal Referral Hospital, 250 Parramatta Road, Homebush West, NSW 2140, Australia BVetnostics, 60 Waterloo Road, North Ryde, NSW 2113, Australia CCentre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, Westmead Hospital, Westmead, NSW 2145, Australia DFaculty of Veterinary Science, The University of Sydney, NSW 2006, Australia ECentre for Veterinary Education, The University of Sydney, NSW 2006, Australia FCorresponding author. Email: [email protected]

Pneumocystis canis is a potential cause of life-threatening to develop fast molecular techniques to diagnose P. canis† interstitial fungal pneumonia in dogs. It is seen almost pneumonia (PCP)7,9 and to determine the underlying exclusively in two canine breeds, miniature Dachshunds immune defect in over-represented breeds through the and Cavalier King Charles Spaniels (CKCS)1. Historically, rapidly advancing field of canine genomics10. Australian veterinarians had a key role in the documentation Pneumocystis spp. are ubiquitous commensals of the respiratory of this entity and its conspicuous breed associations2–4. tract of many mammalian species, including dogs1. This group of Affected Dachshunds and CKCS are likely to have an inher- organisms has the potential to cause life-threatening pneumonia in a ited immunodeficiency that predisposes them to wide range of mammals, including rats, pigs, horses and goats1. with this commensal organism of the and P. jirovecii has the same pathogenic potential in people1. Airborne pharynx1,2,5,6. A high index of suspicion is required to make droplet transmission from subclinically affected normal dogs, often a timely diagnosis and save affected patients, as these dogs transmitted from bitch to pup soon after birth, is suspected of being cope poorly with anaesthesia and other measures to procure the means for transmission1. PCP results from the effects of masses the specimens required to make a definitive diagnosis. of organisms within the alveolar spaces, combined with the asso- Bordetella bronchiseptica Possible co-infection with must – ciated inflammatory response of the host1 6. be considered when determining antimicrobial strategies. Affected dogs occasionally have a previous or concurrent Historically, most canine cases had been reported in young (less – history of generalised demodicosis5,7,8. With early interven- than one-year-old) miniature Dachshunds1 3,6,11. The first docu- tion, affected dogs can be saved, although some require life- mentation of this entity was provided in a paper from the University long therapy to prevent recurrence. The future challenge is of Sydney (UoS) by Farrow and colleagues2, although a brilliant

† The literature refers to Pneumocystis carinii infection of dogs, although recent taxonomic developments suggest this name should be probably restricted to organisms that infect rats. One conference Abstract suggests the name Pneumocystis canis9, although Pneumocystis special form canis may be a safer term in the interim. For the purposes of this article we have used the term P. canis for simplicity.

MICROBIOLOGY AUSTRALIA * MAY 2015 10.1071/MA15026 79 Under the Microscope

(a)(b)

Figure 1. (a) Dorsoventral thoracic radiograph from a one-year-old Cavalier King Charles spaniel with pneumocystosis, revealing a patchy, diffuse, interstitial pattern in all fields. (b) Right lateral thoracic radiograph of the same dog reveals a diffuse interstitial to alveolar pattern, especially in the dorsocaudal lung fields. immunoparasitologist at McMaster Laboratory (Robert J. Love) pro- this was common in the Camden district in the 1970s. A test was vided key immunologic insights.z The disease in Dachshunds was developed for this genetic disease of horses, and nowadays the characterised further by South African veterinarians, with Lobetti condition is hardly encountered, a cogent example of genetic and colleagues adapting human treatment strategies to successfully counselling and preventative veterinary medicine16. manage their canine cases1,11. The best radiological description of In dogs, the nature of the immune defect in Dachshunds and PCP was provided by Kirberger12, although this has been comple- Cavaliers has not been determined at the molecular level. Poor mented by recent advances in imaging such as digital radiology and lymphocyte stimulation (despite normal lymphocyte numbers computed tomography (CT). Although PCP continues to be seen in in peripheral blood) has been documented in miniature Dachs- Dachshunds in South Africa and the USA, the great majority of cases hunds1,2. IgA, IgM and IgG concentrations are subnormal in affected encountered in Australia over the past 20 years have been in adult members of this breed6. Subsequent assessment of CKCS with CKCS, the first detailed report being provided by Paul Canfield pneumocystosis also reveals immunoglobulin deficiencies and de- and colleagues from UoS in 19934. This breed preponderance was creased lymphocyte function5,7. These immunodeficiencies persist then detected in the UK5,13 and subsequently Europe, the USA after resolution of the infection with effective antimicrobial therapy. and Japan7. ‘Cavaliers’ tend to get the disease as young adult Studies of the involvement of adaptive cell-mediated immunity and dogs4,5,13. There may be antecedent or concurrent footprints of innate immunity (including the potential involvement of Toll-like immune deficiency, such as generalised demodicosis5,8. receptors) would be sensible but have not yet been performed.

In people, the disease is best known as a complication of HIV/AIDS, Most dogs with PCP present with respiratory signs, including dys- although it is also seen in transplant recipients and other patients pnoea, tachypnoea, increased breath sounds on thoracic ausculta- receiving immunosuppressive drug regimens. A very well charac- tion or cyanotic mucous membranes. The presence of a cough is terised case cluster in Australia that closed a transplant ward at variable, and this sign is frequently absent. Duration of signs prior to Westmead Hospital was reported by Sharon Chen, Wieland Meyer presentation for veterinary attention can be as long as four weeks1–4. and their ANZMIG colleagues14. In Arabian horses and related Haematologic and serum biochemical abnormalities are non-spe- breeds, foals with autosomal recessive severe combined immuno- cific1. Thoracic radiographs reveal diffuse, bilaterally symmetrically deficiency syndrome (analogous to the like-named SCID condition increased radiodensity of the pulmonary parenchyma, classically of men and mice) typically died of PCP in the neonatal period15, and described as a miliary-interstitial to alveolar pattern (Figure 1)1,12,17.

z The first report of PCP pneumonia in dogs was actually from Germany in 1955. The patient was a nine-week-old sheepdog pup with Pneumocystis forms in its , hilar lymph nodes and myocardium. Sedlmeier, H. and Dahme, E. (1955) Pneumocystis carinii-Infektionbiem Hund. Zentralbl. Allg. Path. 93, 150–155.

80 MICROBIOLOGY AUSTRALIA * MAY 2015 Under the Microscope

Figure 3. A single cyst-like structure (red arrow) suspicious of Pneumocystis spp. ‘cyst’ seen on cytology from a one-year-old Cavalier King Charles Spaniel with pneumocystosis (Diff Quik stain, Figure 2. Thoracic computer tomography scan in the same dog as oil immersion microscopy). Figure 1 reveals a marked, patchy, increased pulmonary opacity throughout all lung lobes with some peripheral consolidation. The Treatment includes appropriate antimicrobial therapy, using intra- ground-glass appearance of the lung parenchyma is quite characteristic of advanced PCP in the dog. venous trimethoprim sulfonamide combinations, and less com- monly nebulised pentamidine, along with appropriate supportive There is often radiological evidence of right-sided heart enlarge- care (e.g. supplementary oxygen, nebulisation, chest physiothera- ment and , and this can be confirmed py). Long-term ventilation of affected dogs is challenging and echocardiographically4. Solitary opacities, cavitary lesions or a beyond the financial limits of most owners. There is no zoonotic non-symmetric radiographic changes are less commonly ob- potential for humans in close contact with a Pneumocystis infected served1,12. Severe long-standing cases may develop emphysema1. dogs, as the organisms in humans and dogs are distinct and no The CT findings in canine PCP are dramatic (Figure 2), providing a evidence of cross-species transmission has been documented. better appreciation of the lung pathology and disclosing regional lymphadenomegaly. As thoughtful and responsible veterinary physicians, we should be banking DNA from all dogs with confirmed and presumptive Diagnosis can be challenging in a veterinary context, with identifi- PCP. The most likely way to eliminate this condition is by harnessing cation of P. canis ‘cysts’ in bronchoalveolar lavage fluid (BAL) the power of canine genomics to detect any underlying genetic or transthoracic fine needle aspirates of lung generally being re- defect, utilising either a genome-wide association study or whole quired. Intact cysts are distinctive, being 5–10 mm in diameter and genome sequencing of ‘Trios’ of affected and closely related dogs10. containing 4–8 dark-staining intracystic bodies (2–3 mm in diameter; It is much better to try and prevent this severe mycosis by screening Figure 3). PCR testing of BAL fluid or lung aspirates is uncommon in the breeding population using a molecular genetic test, rather veterinary laboratories, but should probably be attempted more than to have to treat a severely affected individual. This is where often7,9, as the requirement to perform open lung biopsies to secure vets have an advantage over medics, we can use eugenics! a diagnosis is unnecessarily invasive given the characteristic nature of the imaging findings and the strong breed associations. If BAL References fluid specimens are obtained, they should be subjected to routine 1. Lobetti, R. (2014) Pneumocystosis. In Canine and Feline Infectious Disease bacterial culture as well as microscopy and PCR, as Bordetella (Sykes, J.E., ed.) Saunders, Philadelphia, pp. 689–692. bronchiseptica can be an important co-morbidity in canine PCP 2. Farrow, B.R. et al. (1972) in the dog. J. Comp. Pathol. 82, 447–453. doi:10.1016/0021-9975(72)90044-8 pneumonia. If a BAL is not done and treatment is based on a 3. Copland, J.W. (1974) Canine pneumonia caused by Pneumocystis carinii. Aust. presumptive diagnosis, then doxycycline should be given as well Vet. J. 50, 515–518. doi:10.1111/j.1751-0813.1974.tb14058.x as trimethoprim sulfonamide to cover this possibility. Research is 4. Canfield, P.J. et al. (1993) Pneumocystis pneumonia in a dog. Aust. Vet. Pract. 23, 150–154. currently focused on development of qPCR assays for canine 5. Watson, P.J. et al. (2006) Immunoglobulin deficiency in Cavalier King Charles P. carinii strains, as assays developed for P. jirovecii may not detect Spaniels with Pneumocystis pneumonia. J. Vet. Intern. Med. 20,523–527. P. canis associated with in dogs1. Panfungal PCR with doi:10.1111/j.1939-1676.2006.tb02891.x fi sequence analysis provides an interim molecular diagnostic strate- 6. Lobetti, R. (2000) Common variable immunode ciency in miniature dachshunds affected with Pneumocystis carinii pneumonia. J. Vet. Diagn. Invest. 12,39–45. 7–9 gy, as does genus-specific Pneumocystis PCR assays . doi:10.1177/104063870001200107

MICROBIOLOGY AUSTRALIA * MAY 2015 81 Under the Microscope

7. Hagiwara, Y. et al. (2001) Pneumocystis carinii pneumonia in a Cavalier King in all aspects of veterinary laboratory medicine. He has been – Charles Spaniel. J. Vet. Med. Sci. 63, 349 351. doi:10.1292/jvms.63.349 instrumental in establishing a successful veterinary cytology 8. Furuta, T. et al. (1994) Spontaneous Pneumocystis carinii infection in the dog with naturally acquired generalised demodicosis. Vet. Rec. 134, 423–424. department within Vetnostics over the past decade. His interests doi:10.1136/vr.134.16.423 include the application of advanced diagnostic techniques such as 9. English, K. et al. (2001) DNA analysis of Pneumocystis infecting a Cavalier King immunocytochemistry and flow cytometry in veterinary cytology Charles spaniel. J. Eukaryot. Micro. 48, 106S. doi:10.1111/j.1550-7408.2001. tb00471.x and haematology as well as establishing protocols for PCR testing 10. Karlsson, E.K. and Lindblad-Toh, K. (2008) Leader of the pack: gene mapping of some infectious agents on cytology specimens. in dogs and other model organisms. Nat. Rev. Genet. 9, 713–725. doi:10.1038/ nrg2382 Catriona Halliday is the Senior Scientist in charge of the Clinical 11. Lobetti, R.G. et al. (1996) Pneumocystis carinii in the miniature dachshund: case report and literature review. J. Small Anim. Pract. 37,280–285. doi:10.1111/ Mycology Reference Laboratory at Westmead Hospital where she j.1748-5827.1996.tb02380.x has been working for over 14 years. Her research interests have 12. Kirberger, R.M. and Lobetti, R.G. (1998) Radiographic aspects of Pneumocystis focused on the development and implementation of culture carinii pneumonia in the miniature Dachshund. Vet. Radiol. Ultrasound 39, 313–317. doi:10.1111/j.1740-8261.1998.tb01612.x independent tests to aid in the rapid diagnosis of invasive fungal 13. Ramsey, I.K. et al. (1997) Pneumocystis carinii pneumonia in two Cavalier King infections, in particular invasive . Charles spaniels. Vet. Rec. 140, 372–373. doi:10.1136/vr.140.14.372 14. Phipps, L.M. et al. (2011) Nosocomial Pneumocystis jirovecii pneumonia: lessons Dr Mark Krockenberger graduated from the University of from a cluster in kidney transplant recipients. Transplantation 92, 1327–1334. doi:10.1097/TP.0b013e3182384b57 Sydney in 1993, and has worked in dairy and small animal practice 15. Perryman, L.E. (2004) Molecular pathology of severe combined immunodeficien- in Australia and United Kingdom. After completing a PhD on – cy in mice, horses, and dogs. Vet. Pathol. 41,95 100. doi:10.1354/vp.41-2-95 cryptococcosis in koalas in 2001, Mark accepted a Lecturer in 16. Ainsworth, D.M. et al. (1993) Recognition of Pneumocystis carinii in foals with respiratory distress. Equine Vet. J. 25, 103–108. doi:10.1111/j.2042-3306.1993. Veterinary pathology at the University of Sydney. After rising tb02917.x through the ranks, he is now an Associate Professor and 17. Cabañes, F.J. et al. (2000) Pneumocystis carinii pneumonia in a Yorkshire terrier head of the Diagnostic Laboratory. His research includes the dog. Med. Mycol. 38, 451–453. doi:10.1080/714030971 host–pathogen–environment interactions of cryptococcosis, diseases of koalas and diseases of Australian wildlife. He is Biographies especially interested in the pathogenesis of fungal disease, Elizabeth Ralph is a veterinarian and has recently finished her including in laboratory models. residency in small animal internal medicine and is well on the way to Richard Malik is a consultant in small animal medicine that has a her goal of becoming a small animal internal medicine specialist. special interest in infectious diseases of companion animals. He Elizabeth has also worked with the Department of Primary Indus- is particularly interested in viral diseases of cats, fungal diseases tries during the Equine Influenza outbreak in 2007 and with her local especially those caused by Cryptococcus species, mycobacteria, government as a member of the Companion Animal Advisory saprophytic pathogens such as Burholderia, Prototheca and Committee. Her professional interests include small animal internal Pythium and most recently parasitic diseases. Richard works for medicine, especially immune-mediated disease. the Centre for Veterinary Education where he facilitates feline George Reppas is a specialist veterinary pathologist at Vetnostics distance education programs and develops life-long learning strat- (the veterinary division of Laverty Pathology NSW) and is involved egies for vets in practice.

82 MICROBIOLOGY AUSTRALIA * MAY 2015